Blood is composed of plasma and cellular constituents and plays a prominent role in several key physiological systems of the human body, including immunology, hemostasis, and tissue oxygenation. Movement of the respiratory gases oxygen (O2) and carbon dioxide (CO2) is the chief function of erythrocytes or red blood cells. This facilitated movement of O2 and CO2 is carried out by hemoglobin, an iron-containing, multi-subunit protein contained within red blood cells. The necessity for encapsulating hemoglobin within the red blood cell is twofold. First, hemoglobin has very specific binding parameters to ensure delivery of these critical molecules to the proper sites. By regulating the concentration of cofactors and free ions, the red blood cell provides the proper environment for optimum uptake and release of O2. Secondly, free heme has toxicity effects on both the renal and hepatic systems and can lead to conditions such as hemoglobinuria.
The red blood cell structural components which confine the hemoglobin are comprised of the membrane bilayer and the cytoskeleton. The membrane itself contains choline and amino phospholipids, cholesterol, and integral membrane proteins. Along the inner surface of the membrane lies the cytoskeleton. This mesh-like skeleton is composed of long, filamentous spectrin molecules joined together at foci of F-actin and protein 4.1 complexes. This cytoskeletal mesh is linked to the membrane through protein-protein interactions such as the ankyrin/protein 4.2 mediated connection of spectrin and the integral membrane protein Band 3, and possibly via direct protein-lipid interactions. Through these connections, the cytoskeleton provides the red blood cell its stability and shape. In the absence of proper binding within the cytoskeletal mesh or the connection of the cytoskeleton to the membrane, a critically weakened red blood cell results. Several human diseases have been identified stemming from either an absence of a specific protein or a defective protein interaction. Most of these defects lead to morphologically abnormal red blood cells and severe hemolytic anemias.
A wide variety of injuries and medical procedures require the transfusion of whole blood or a variety of blood components. Blood transfusions are routinely used to increase oxygen delivery capacity and circulatory volume in patients. Safe, quick, and easy access to transfusable red blood cell units is not only important for trauma victims with massive blood loss, but also for patients undergoing elective surgery or who have diseases, such as several types of hereditary hemolytic anemias, which result in a loss of circulating red blood cells. The ability to store red blood cells for extended time periods ensures that a supply of transfusable red blood cells will be available when needed. Potential uses include storage of unique serotypes, units intended for autologous transfusion and stockpiling general blood types for emergency situations. Limitations in the current storage methodologies have led, in part, to occasional shortages of blood supply, resulting in postponement of elective surgeries and calls for donations from blood banks and hospitals.
Currently, two methods are approved for extended storage of red blood cells. The majority of red blood cell units are stored in citrate phosphate-dextrose at 4° C. For example, when donor blood is received at a processing center, erythrocytes are separated and stored by various methods, generally as a unit of packed erythrocytes having a volume of from 200 to 300 ml and a hematocrit value of 70 to 90. However, due to metabolic depletion and subsequent physical degradation, these cells may be stored no longer than 42 days. Furthermore, while 70% of these stored cells remain in circulation following transfusion, true O2 delivery capability has not been demonstrated.
During storage, human red blood cells undergo morphological and biochemical changes, including decreases in the cellular level of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG), changes in cellular morphology, and progressive hemolysis. The concentration of ATP, after a brief initial rise, progressively declines to between 30 and 40% of its initial level after six weeks of storage. Morphological changes occur during storage, ultimately leading to the development of spicules which can bud off as vesicles, radically changing the surface-to-volume ratio of the cells and their ability to deform on passing through narrow channels. The fluidity of the cell membrane of red cells, which is essential for the passage of red cells through the narrow channels in the spleen and liver, is loosely correlated with the level of ATP. The concentration of ATP and the morphology of red cells serve as indicators of the suitability of stored cells for transfusion.
The second method is frozen storage at −80° C. using 40% (w/v) glycerol as a cryoprotectant. However, this additive penetrates the membranes of many biological cells and possesses unwanted properties when infused into humans. While this method can be used for up to 10 years of storage, the glycerol must be washed out prior to transfusion. The washing procedure utilizes costly equipment and is time and labor intensive. Additionally, a significant level of hemolysis (10-15% typically) occurs during the wash procedure. Finally, since this procedure is also considered to compromise the sterility of the red blood cell units, post-wash storage is significantly limited. Consequently, this method of storage is only used for blood with extremely rare factor types and autologous and directed donation units which will not be used within a 42 day period. As of 1992, the most recent data available, approximately 14% (nearly 2 million units) of the available supply of transfusable red blood cells were discarded. The ability to cryopreserve red blood cells without the added expense and time of washing, or at least reducing the number of washing cycles necessary, would enable salvage of a significant percentage of these discarded units, thereby providing a further buffer against blood product is shortages.
Current theories of red blood cell cryopreservation consider ice formation and propagation to be the primary, if not the sole factor affecting cell recovery and viability following storage. Consequently, investigation of methods to cryopreserve red blood cells only consider slight variations of traditional cryoprotectant methodologies. Prior methods for the preservation, storage, and transfusion of red blood cells are explained in Horn, Sputtek, Standl, Rudolf, Kuhnl, and Esch, Transfusion of Autologous, Hydroxyethyl Starch-Cryopreserved Red Blood Cells, Anesth. Analg. 1997; 85; 739-745. However, temperature-induced modulation of cellular biochemistry is a well recognized phenomenon by experts in this field. The descriptions herein provide methods of utilizing the red blood cell biochemistry to overcome cold-induced imbalances which would normally lead to cellular hemolysis following cryopreservation. Through these methods, the level of non-specific cryoprotection against ice formation may be reduced.
Two general procedures are currently available for the frozen preservation of living cells in terms of the cryoprotectant used. One utilizes the addition of high concentrations of low molecular weight penetrant solutes. Due to toxicity and osmotic problems, specialized equipment and techniques are required to remove the solutes following thawing. The second utilizes high molecular weight polymers which do not enter the cells. Post-thaw processing in this case is logistically simpler but storage must be at very low temperature, typically in liquid nitrogen. The formation of ice, which is a prime concern in any method of cell cryopreservation, is initiated by ice crystal nuclei. As the temperature falls and water crystallizes to ice, the cell is progressively dehydrated and at some point cell injury results. The nature of the dehydration injury is believed to be the result of membrane stresses leading to membrane rupture. This form of injury can be prevented by the addition of solutes at a multi-molar concentration so that the amount of ice formed is insufficient to result in damaging cell dehydration.
For many years it has been known that certain molecules may be cryoprotective. It is necessary for these penetrating cryoprotectants to reduce the amount of extracellular ice formed and thereby reduce cell dehydration, while at the same time increasing the intracellular concentration to make intracellular crystallization less likely. Such a solute, to be useful, must be non-toxic at high concentration and must freely penetrate the cell. Both glycerol and dimethylsulfoxide (DMSO) have been used for this purpose. Glycerol is remarkably non-toxic at high concentrations but penetrates cell membranes slowly and is therefore difficult to introduce and remove. Dimethylsulfoxide penetrates rapidly but becomes increasingly toxic as concentrations exceed 1M (about 7%). Large molecular weight polymers, such as polyvinylpyrrolidone (PVP), dextran, and more recently hydroxyethyl starch (HES), do not enter the cell, and the mechanism by which they confer cryoprotection has been the subject of speculation.
As stated above, in order to prolong the storage of red blood cells it is necessary to store the cells or treat them in some manner that prevents a decline in ATP, and, if possible, 2,3-diphosphoglycerate (2,3-DPG), in addition to protection against ice crystal damage. Typically, such solutions contain phosphate, glucose, and adenine which function to prolong shelf-life by maintaining the level of ATP in the cells. In addition, glycolytic activity is enhanced in red blood cells if the intracellular pH measured at 4° C. is about 7.4 The effective osmolality of the suspending solution is another factor of importance in extending red cell storage time. It has been shown that hypotonically induced increases in mean cell volume substantially reduce hemolysis and improves red cell morphology during storage. Although the mechanism has not been proven, it is possible that osmotic swelling increases cell surface tension, thereby opposing the shape changes usually associated with stored red cells. Although the hypotonicity of the additive solution is limited by the danger of hemolysis during the addition of the solution, red cells, which are normally bi-concave disks, can swell to nearly twice their normal volume at an external osmolality of approximately 170 mOsm before they hemolyze. If the additive solution is too hypotonic, the red cells will burst (hemolyze). As a result, solutions that are too hypotonic cannot be used. While maintenance of ATP and 2,3-DPG are generally thought of with reference to 4° C. storage, maintaining the concentration of these metabolites is important for post-thaw storage. For the effects on ATP levels, hemolysis, potassium leakage, and shedding of microvesicles, on the maintenance and storage of red blood cells, see Greenwalt, Rugg, and Dumaswala, The Effect of Hypotonicity Glutamine, and Glycine on Red Cell Preservation, Transfusion 1997; 37; 269-276.
The goal of extended red blood cell storage is to maintain the metabolic and morphologic characteristics so that the in vivo parameters (oxygen delivery capacity and circulatory half-life) are comparable to fresh, non-frozen red blood cells. Additionally, the level of hemolysis during the storage cycle must remain within acceptable levels to allow direct transfusion of the thawed red blood cell unit without complications from hemoglobin toxicity. These goals can be accomplished using the method of this invention. The method described herein provides the advantages of both currently approved storage methods—extended storage time in the frozen state and immediate availability as in refrigerated storage.
Hospitals and blood banks would greatly benefit from a directly-transfusable frozen red blood cell product. Most current investigational approaches to accomplish this attempt to merely replace glycerol with a non-toxic, transfusable cryoprotectant. However, results have shown that most cryoprotectants need to be used at concentrations which may not be transfused or their protective qualities are not enough to maintain hemolysis at acceptable levels for transfusion. Additionally, most procedures require storage at −193° C. in liquid nitrogen vapor. This would be expensive and difficult to incorporate into current blood banking procedures, making extended storage at temperatures below −80° C. impractical.
The invention described herein addresses the noted problems of storage, morphological changes, metabolic changes, and long term functional effectiveness of red blood cells. Further, the present invention achieves unexpected and surprising results in the preservation of red blood cells through treating with the compositions and methods of this invention and which previously would have been considered impossible.